PH-Triggered Reversible Multiple Protein-Polymer Conjugation Based on Molecular Recognition

Polymer conjugation for protein-based therapeutics has been developed extensively, but it still suffers from conjugation leading to decrease in protein activity and generates complexes with limited diversity due to general classical systems only incorporating one protein per each complex. Here we introduce a site-specific noncovalent protein-polymer conjugation, which can reduce the heterogeneity of the conjugates without disrupting protein function, while allowing for the modulation of binding affinity and stability, affecting the pH dependent binding of the number of proteins per polymer. We compared classical one protein-polymer conjugates with multiple protein-polymer conjugates using His-tagged enhanced yellow fluorescence protein (His6-eYFP) and metal-coordinated tris-nitrilotriacetic acid (trisNTA-Men+) in a site-specific way. trisNTA-Men+-His6 acts as a reversible linker with pH-triggered release of functional protein from the trisNTA-functionalized copolymers. The nature of the selected Men+ and number of available trisNTA-Men+ on poly(N-isopropylacrylamide-co-tris-nitrilotriacetic acid acrylamide) (PNTn) copolymers enables predictable modulation of the conjugates binding affinity (0.09-1.35 μM), stability, cell toxicity, and pH responsiveness. This represents a promising platform that allows direct control over the properties of multiple protein-polymer conjugates compared to the classical single protein-polymer conjugates.